Device and method for determining analyte levels

ABSTRACT

Devices and methods for determining analyte levels are described. The devices and methods allow for the implantation of analyte-monitoring devices, such as glucose monitoring devices, that result in the delivery of a dependable flow of blood to deliver sample to the implanted device. The devices comprise a unique microarchitectural arrangement in the sensor region that allows accurate data to be obtained over long periods of time.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.08/811,473, filed on Mar. 4, 1997, which issued as U.S. Pat. No.6,001,067 n Dec. 14, 1999.

FIELD OF THE INVENTION

The present invention relates generally to devices and methods fordetermining analyte levels, and, more particularly, to implantabledevices and methods for monitoring glucose levels in a biological fluid.

BACKGROUND OF THE INVENTION

The continuous measurement of substances in biological fluids is ofinterest in the control and study of metabolic disorders. Electrodesystems have been developed for this purpose whereby an enzyme-catalyzedreaction is monitored (e.g., by the changing concentrations of reactantsor products) by an electrochemical sensor. In such electrode systems,the electrochemical sensor comprises an electrode with potentiometric oramperometric function in close contact with a thin layer containing anenzyme in dissolved or insoluble form. Generally, a semipermeablemembrane separates the thin layer of the electrode containing the enzymefrom the sample of biological fluid that includes the substance to bemeasured.

Electrode systems that include enzymes have been used to convertamperometrically inactive substances into reaction products which areamperometrically active. For example, in the analysis of blood forglucose content, glucose (which is relatively inactive amperometrically)may be catalytically converted by the enzyme glucose oxidase in thepresence of oxygen and water to gluconic acid and hydrogen peroxide.Tracking the concentration of glucose is possible since for everyglucose molecule converted a proportional change in either oxygen orhydrogen peroxide sensor current will occur [U.S. Pat. Nos. 4,757,022and 4,994,167 to Shults et al., both of which are hereby incorporated byreference]. Hydrogen peroxide is anodically active and produces acurrent which is proportional to the concentration of hydrogen peroxide,which is directly related to the concentration of glucose in the sample.[Updike et al., Diabetes Care, 11:801-807 (1988)].

Despite recent advances in the field of implantable glucose monitoringdevices, presently used devices are unable to provide data safely andreliably for long periods of time (e.g., months or years) [See, e.g.,Moatti-Sirat et al., Diabetologia 35:224-30 (1992)]. For example, Armouret al., Diabetes 39:1519-26 (1990), describes a miniaturized sensor thatis placed intravascularly, thereby allowing the tip of the sensor to bein continuous contact with the blood. Unfortunately, probes that areplaced directly into the vasculature put the recipient at risk forthrombophlebosis, thromboembolism, and thrombophlebitis.

Currently available glucose monitoring devices that may be implanted intissue (e.g., subcutaneously) are also associated with severalshortcomings. For example, there is no dependable flow of blood todeliver sample to the tip of the probe of the implanted device.Similarly, in order to be effective, the probe must consume some oxygenand glucose, but not enough to perturb the available glucose which it isintended to measure; subcutaneously implanted probes often reside in arelatively stagnant environment in which oxygen or glucose depletionzones around the probe tip may result in erroneously low measuredglucose levels. Finally, the probe may be subject to “motion artifact”because the device is not adequately secured to the tissue, thuscontributing to unreliable results. Partly because of these limitations,it has previously been difficult to obtain accurate informationregarding the changes in the amounts of analytes (e.g., whether bloodglucose levels are increasing or decreasing); this information is oftenextremely important, for example, in ascertaining whether immediatecorrective action is needed in the treatment of diabetic patients.

There is a need for a device that accurately and continuously determinesthe presence and the amounts of a particular analyte, such as glucose,in biological fluids. The device should be easy to use, be capable ofaccurate measurement of the analyte over long periods of time, andshould not readily be susceptible to motion artifact.

SUMMARY OF THE INVENTION

The present invention relates generally to devices and methods fordetermining analyte levels, and, more particularly, to implantabledevices and methods for monitoring glucose levels in a biological fluid.

The devices and methods of the present invention allow for theimplantation of analyte-monitoring devices such as glucose monitoringdevices that result in a dependable flow of blood to deliver sample tothe implanted device at a concentration representative of that in thevasculature. Moreover, the devices of the present invention becomesecured within the tissue of the subject, thereby greatly reducing oreliminating the phenomenon of “motion artifact”. In addition, thedevices of the present invention utilize materials that eliminate orsignificantly delay environmental stress cracking at the sensorinterface, resulting in the ability to obtain accurate, long-term data.

These effects result, in part, from the use of materials that enhancethe formation of a foreign body capsule (FBC). Previously, FBC formationhas been viewed as being adverse to sensor function, and researchershave attempted to minimize FBC formation (see, e.g., U.S. Pat. No.5,380,536 to Hubbell et al.). However, the methods and devices of thepresent invention utilize specific materials and microarchitecture thatelicit a type of FBC that does not hamper the generation of reliabledata for long periods. The devices of the present invention are capableof accurate operation in the approximately 37° C., low pO₂, environmentcharacteristic of living tissue for extended lengths of time (e.g.,months to years).

The electrode-membrane region of the devices of the present inventioncomprises a unique microarchitectural arrangement. In preferredembodiments, the electrode surfaces are in contact with (or operablyconnected with) a thin electrolyte phase, which in turn is covered by anenzyme membrane that contains an enzyme, e.g., glucose oxidase, and apolymer system. A bioprotective membrane covers this enzyme membranesystem and serves, in part, to protect the sensor from external forcesand factors that may result in environmental stress cracking. Finally,an angiogenic layer is placed over the bioprotective membrane and servesto promote vascularization in the sensor interface region. It is to beunderstood that other configurations (e.g., variations of that describedabove) are contemplated by the present invention and are within thescope thereof.

The present invention contemplates a biological fluid measuring device,comprising a) a housing comprising electronic circuit means and at leasttwo electrodes operably connected to the electronic circuit means; andb) a sensor means operably connected to the electrodes of the housing,the sensor means comprising i) a bioprotective membrane, and ii) anangiogenic layer, the angiogenic layer positioned more distal to thehousing than the bioprotective membrane. In particular embodiments, thebioprotective membrane is substantially impermeable to macrophages. Insome embodiments, the bioprotective membrane comprises pores havingdiameters ranging from about 0.1 micron to about 1.0 micron. In certainembodiments, the bioprotective membrane comprisespolytetrafluoroethylene, and in particular embodiments, the angiogeniclayer also comprises polytetrafluoroethylene.

Particular embodiments of the biological fluid measuring device furthercomprise c) means for securing the device to biological tissue, thesecuring means associated with the housing. In some embodiments, thesecuring means comprises a polyester velour jacket. In preferredembodiments, the securing means covers the top surface (e.g., the topmember or the top member sheath, as described further below) and aportion of the sensor interface; it should be noted that the securingmeans generally should not cover the entire sensor interface, as thiswould interfere with the ability of blood vessels to deliver sample tothe biological fluid measuring device. In preferred embodiments, thesecuring means comprises poly(ethylene terephthalate).

In further embodiments, the sensor means of the biological fluidmeasuring device further comprises means for determining the amount ofglucose in a biological sample. In some embodiments, the glucosedetermining means comprises a membrane containing glucose oxidase, theglucose oxidase-containing membrane positioned more proximal to thehousing than the bioprotective membrane. In additional embodiments, thehousing further comprises means for transmitting data to a locationexternal to the device (e.g., a radiotelemetry device).

The present invention also contemplates a device for measuring glucosein a biological fluid, comprising a) a housing comprising electroniccircuit means and at least one electrode operably connected to theelectronic circuit means; and b) a sensor means operably connected tothe electrode of the housing, the sensor means comprising i) means fordetermining the amount of glucose in a biological sample, the glucosedetermining means operably associated with the electrode, ii) abioprotective membrane, the bioprotective membrane positioned moredistal to the housing than the glucose determining means andsubstantially impermeable to macrophages, and iii) an angiogenic layer,the angiogenic layer positioned more distal to the housing than thebioprotective membrane.

In particular embodiments, the glucose determining means comprises amembrane containing glucose oxidase. In some embodiments, the angiogeniclayer comprises polytetrafluoroethylene.

In some embodiments, the pores of the bioprotective membrane havediameters ranging from about 0.1 micron to about 1.0 micron, while inother embodiments the pores have diameters ranging from about 0.2 micronto about 0.5 micron. In certain embodiments, the bioprotective membranecomprises polytetrafluoroethylene.

Still other embodiments further comprise c) means for securing thedevice to biological tissue, the securing means associated with thehousing. In particular embodiments, the securing means comprisespoly(ethylene terephthalate). Additional embodiments comprise means fortransmitting data to a location external to the device; in someembodiments, the data transmitting means comprises a radiotelemetricdevice.

The present invention also contemplates a method for monitoring glucoselevels, comprising a) providing i) a host, and ii) a device comprising ahousing and means for determining the amount of glucose in a biologicalfluid; and b) implanting the device in the host under conditions suchthat the device measures the glucose accurately for a period exceeding90 days. In some embodiments, the device measures glucose accurately fora period exceeding 150 days, while in other embodiments, the devicemeasures glucose accurately for a period exceeding 360 days.

The present invention also contemplates a method of measuring glucose ina biological fluid, comprising a) providing i) a host, and ii) a devicecomprising a housing and means for determining the amount of glucose ina biological fluid, the glucose determining means capable of accuratecontinuous glucose sensing; and b) implanting the device in the hostunder conditions such that the continuous glucose sensing begins betweenapproximately day 2 and approximately day 25. In some embodiments, thecontinuous glucose sensing begins between approximately day 3 andapproximately day 21. In particular embodiments, the implanting issubcutaneous.

The devices of the present invention allow continuous informationregarding, for example, glucose levels. Such continuous informationenables the determination of trends in glucose levels, which can beextremely important in the management of diabetic patients.

DEFINITIONS

In order to facilitate an understanding of the present invention, anumber of terms are defined below.

The term “accurately” means, for example, 95% of measured values within25% of the actual value as determined by analysis of blood plasma,preferably within 15% of the actual value, and most preferably within 5%of the actual value. It is understood that like any analytical device,calibration, calibration check and recalibration are required for themost accurate operation of the device.

The term “analyte” refers to a substance or chemical constituent in abiological fluid (e.g., blood or urine) that can be analyzed. Apreferred analyte for measurement by the devices and methods of thepresent invention is glucose.

The terms “sensor interface,” “sensor means,” and the like refer to theregion of a monitoring device responsible for the detection of aparticular analyte. For example, in some embodiments of a glucosemonitoring device, the sensor interface refers to that region wherein abiological sample (e.g., blood or interstitial fluid) or a portionthereof contacts (directly or after passage through one or moremembranes or layers) an enzyme (e.g., glucose oxidase); the reaction ofthe biological sample (or portion thereof) results in the formation ofreaction products that allow a determination of the glucose level in thebiological sample. In preferred embodiments of the present invention,the sensor means comprises an angiogenic layer, a bioprotective layer,an enzyme layer, and an electrolyte phase (i.e., a free-flowing liquidphase comprising an electrolyte-containing fluid [described furtherbelow]). In some preferred embodiments, the sensor interface protrudesbeyond the plane of the housing.

The terms “operably connected,” “operably linked,” and the like refer toone or more components being linked to another component(s) in a mannerthat allows transmission of, e.g., signals between the components. Forexample, one or more electrodes may be used to detect the amount ofanalyte in a sample and convert that information into a signal; thesignal may then be transmitted to electronic circuit means (i.e., theelectrode is “operably linked” to the electronic circuit means), whichmay convert the signal into a numerical value in the form of knownstandard values.

The term “electronic circuit means” refers to the electronic circuitrycomponents of a biological fluid measuring device required to processinformation obtained by a sensor means regarding a particular analyte ina biological fluid, thereby providing data regarding the amount of thatanalyte in the fluid. U.S. Pat. No. 4,757,022 to Shults et al.,previously incorporated by reference, describes suitable electroniccircuit means (see, e.g., FIG. 7); of course, the present invention isnot limited to use with the electronic circuit means described therein.A variety of circuits are contemplated, including but not limited tothose circuits described in U.S. Pat. Nos. 5,497,772 and 4,787,398,hereby incorporated by reference.

The terms “angiogenic layer,” “angiogenic membrane,” and the like referto a region, membrane, etc. of a biological fluid measuring device thatpromotes and maintains the development of blood vessels microcirculationaround the sensor region of the device. As described in detail below,the angiogenic layer of the devices of the present invention may beconstructed of membrane materials alone or in combination such aspolytetrafluoroethylene, hydrophilic polyvinylidene fluoride, mixedcellulose esters, polyvinyl chloride, and other polymers including, butnot limited to, polypropylene, polysulphone, and polymethacrylate.

The phrase “positioned more distal” refers to the spatial relationshipbetween various elements in comparison to a particular point ofreference. For example, some embodiments of a biological fluid measuringdevice comprise both a bioprotective membrane and an angiogeniclayer/membrane. If the housing of the biological fluid measuring deviceis deemed to be the point of reference and the angiogenic layer ispositioned more distal to the housing than the bioprotective layer, thenthe bioprotective layer is closer to the housing than the angiogeniclayer.

The terms “bioprotective membrane,” “bioprotective layer,” and the likerefer to a semipermeable membrane comprised of protective biomaterialsof a few microns thickness or more which are permeable to oxygen andglucose and are placed over the tip of the sensor to keep the whiteblood cells (e.g., tissue macrophages) from gaining proximity to andthen damaging the enzyme membrane. In some embodiments, thebioprotective membrane has pores (typically from approximately 0.1 toapproximately 1.0 micron). In preferred embodiments, a bioprotectivemembrane comprises polytetrafluoroethylene and contains pores ofapproximately 0.4 microns in diameter. Pore size is defined as the poresize provided by the manufacturer or supplier.

The phrase “substantially impermeable to macrophages” means that few, ifany, macrophages are able to cross a barrier (e.g., the bioprotectivemembrane). In preferred embodiments, fewer than 1% of the macrophagesthat come in contact with the bioprotective membrane are able to cross.

The phrase “means for securing said device to biological tissue” refersto materials suitable for attaching the devices of the present inventionto, e.g., the fibrous tissue of a foreign body capsule. Suitablematerials include, but are not limited to, poly(ethylene terephthalate).In preferred embodiments, the top of the housing is covered with thematerials in the form of surgical grade fabrics; more preferredembodiments also contain material in the sensor interface region (seeFIG. 1B).

The phrase “means for determining the amount of glucose in a biologicalsample” refers broadly to any mechanism (e.g., enzymatic ornon-enzymatic) by which glucose can be quantitated. For example, someembodiments of the present invention utilize a membrane that containsglucose oxidase that catalyzes the conversion of glucose to gluconate:Glucose+O₂→Gluconate+H₂O₂. Because for each glucose molecule convertedto gluconate, there is a proportional change in the co-reactant O₂ andthe product H₂O₂, one can monitor the current change in either theco-reactant or the product to determine glucose concentration.

The phrase “means for transmitting data to a location external to saiddevice” refers broadly to any mechanism by which data collected by abiological fluid measuring device implanted within a subject may betransferred to a location external to the subject. In preferredembodiments of the present invention, radiotelemetry is used to providedata regarding blood glucose levels, trends, and the like. The terms“radiotelemetry,” “radiotelemetric device,” and the like refer to thetransmission by radio waves of the data recorded by the implanted deviceto an ex vivo recording station (e.g., a computer), where the data isrecorded and, if desired, further processed (see, e.g., U.S. Pat. Nos.5,321,414 and 4,823,808, hereby incorporated by reference; PCT PatentPublication WO 9422367).

The term “host” refers to both humans and animals.

The phrase “continuous glucose sensing” refers to the period in whichmonitoring of plasma glucose concentration is continuously carried out.More specifically, at the beginning of the period in which continuousglucose sensing is effected, the background sensor output noisedisappears, and the sensor output stabilizes (e.g., over several days)to a long-term level reflecting adequate microcirculatory delivery ofglucose and oxygen to the tip of the sensor (see FIG. 2). Though anunderstanding of this effect is not required in order to practice thepresent invention, it is believed to be due to adequately vascularizedforeign body capsule tissue in consistent contact with the sensorinterface of the blood glucose monitoring device. Failure of adequatevascularization or consistent contact of tissue with sensor will resultin failure of continuous glucose sensing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a cross-sectional drawing of one embodiment of animplantable analyte measuring device of the present invention.

FIG. 1B depicts a cross-sectional exploded view of the sensor interfacedome of FIG. 1A.

FIG. 1C depicts a cross-sectional exploded view of theelectrode-membrane region of FIG. 1B detailing the sensor tip and thefunctional membrane layers.

FIG. 2 graphically depicts glucose levels as a function of the number ofdays post-implant.

FIG. 3 graphically depicts a correlation plot (days 21 to 62) of aglucose infusion study with one device of the present invention.

FIG. 4 depicts a typical response to in vitro calibration to glucose ofa device of the present invention.

FIGS. 5A, 5B, and 5C graphically depict three in vivo sensor responsecurves plotted in conjunction with the reference blood glucose valuesfor one device of the present invention at post-implant times of 25, 88,and 109 days.

FIG. 6 graphically depicts sensor glucose versus reference glucose forone device of the present invention using the single set of calibrationfactors from day 88 of FIG. 5B.

DESCRIPTION OF THE INVENTION

The present invention relates generally to devices and methods fordetermining analyte levels, and, more particularly, to implantabledevices and methods for monitoring glucose levels in a biological fluid.In a preferred embodiment, the device and methods of the presentinvention are used to determine the level of glucose in a subject, aparticularly important measurement for individuals having diabetes.

Although the description that follows is primarily directed at glucosemonitoring devices and methods for their use, the devices and methods ofthe present invention are not limited to glucose measurement. Rather,the devices and methods may be applied to detect and quantitate otheranalytes present in biological fluids (including, but not limited to,amino acids and lactate), especially those analytes that are substratesfor oxidase enzymes [see, e.g., U.S. Pat. No. 4,703,756 to Gough et al.,hereby incorporated by reference]. Moreover, the devices and methods ofthe present invention may be utilized to present components ofbiological fluids to measurement methods which are not enzyme-based,including, but not limited to, those based on surface plasmon resonance,surface acoustic waves, optical absorbance in the long wave infraredregion, and optical rotation of polarized light.

I. Nature of the Foreign Body Capsule

Probes that are implanted (e.g., subcutaneously) into tissue will almostalways elicit a foreign body capsule (FBC) as part of the body'sresponse to the introduction of a foreign material. Though a preciseunderstanding of the nature of a FBC is not required in order topractice the present invention, generally speaking, upon implantation ofa glucose sensor, there is initially an acute inflammatory reaction(which includes invasion of tissue macrophages), followed by building offibrotic tissue. A mature capsule (i.e., the FBC) comprising primarilyavascular fibrous tissue forms around the device [Woodward, DiabetesCare, 5:278-281 (1982)]. Although fluid is frequently found within thecapsular space between the sensor and the capsule, levels of analytes(e.g., glucose and oxygen) within the fluid often do not mimic levels inthe body's vasculature, making accurate measurement difficult. Example 4below describes typically identifiable phases in FBC formation asreflected by response of an implanted glucose sensor.

In general, the formation of FBCs has precluded the collection ofreliable, continuous information because they isolate the sensor of theimplanted device from biological fluids, fully equilibrated with atleast the low molecular weight components found in the circulation.Similarly, the composition of FBCs has prevented stabilization of theimplanted device, contributing to motion artifact that rendersunreliable results. Thus, conventionally, it has been the practice ofthose skilled in the art to attempt to minimize FBC formation by, forexample, using a short lived needle geometry or sensor coatings tominimize the foreign body reaction.

In contrast to the prior art, the teachings of the present inventionrecognize that FBC formation is the dominant event surrounding long termimplantation of any sensor and must be orchestrated to support ratherthan hinder or block sensor performance. For example, sensors often donot perform well until the FBC has matured sufficiently to provideingrowth of well attached tissue bearing a rich supply of capillariesdirectly to the surface of the sensor. This maturation process takes atleast several days and, when initiated according to the presentinvention, is a function of biomaterial and host factors which initiateand modulate angiogenesis, and promote and control fibrocyte ingrowth.The present invention contemplates the use of particular materials topromote angiogenesis adjacent to the sensor interface (also referred toas the electrode-membrane region, described below) and to anchor thedevice within the FBC.

II. The Implantable Glucose Monitoring Devices of the Present Invention

The present invention contemplates the use of a uniquemicroarchitectural organization around the sensor interface of animplantable device. Moreover, the present invention contemplates the useof materials covering all or a portion of the device to assist in thestabilization of the device following implantation. However, it shouldbe pointed out that the present invention does not require a devicecomprising particular electronic components (e.g., electrodes,circuitry, etc). Indeed, the teachings of the present invention can beused with virtually any monitoring device suitable for implantation (orsubject to modification allowing implantation); suitable devicesinclude, but are not limited, to those described in U.S. Pat. Nos.4,703,756 and 4,994,167 to Shults et al.; U.S. Pat. No. 4,703,756 toGough et al., and U.S. Pat. No. 4,431,004 to Bessman et al.; thecontents of each being hereby incorporated by reference, and Bindra etal., Anal. Chem. 63:1692-96 (1991).

In the discussion that follows, an example of an implantable device thatincludes the features of the present invention is first described.Thereafter, the specific characteristics of, for example, the sensorinterface contemplated by the present invention will be described indetail.

Generally speaking, the implantable devices contemplated for use withthe present invention are oval shaped; of course, devices with othershapes may also be used with the present invention. The sample deviceincludes a housing having an upper portion and a lower portion whichtogether define a cavity. FIG. 1A depicts a cross-sectional drawing ofone embodiment of an implantable measuring device. Referring to FIG. 1A,the device comprises a main housing (also referred to as casing orpackaging) consisting of a bottom member 1 with upwardly angledprojecting extensions along its perimeter. The four downwardlyprojecting extensions of a similarly-shaped top member 2 engage theupwardly projecting extensions of the bottom member 1. As indicated inFIG. 1A, there is an aperture in top member 2 that allows for protrusionof the convexly curved sensor interface dome 30. Preferred embodimentsof the present invention entail such a protrusion of the sensorinterface dome 30; in some embodiments, though a precise understandingof the effect of the protrusion is not required in order to practice thepresent invention, the protrusion is believed to assist in the formationof vasculature in the sensor interface dome 30 region, and hencepresentation of sample to the electrodes.

In certain embodiments, a top member sheath 4 covers the top member 2;like the top member 2, the top member sheath 4 has an aperture whichallows the sensor interface dome 30, which has a curvature greater thanthat of the surrounding region, to protrude therethrough. As indicatedin detail in FIG. 1B, the top member sheath 4 angles upward as itapproaches the aperture, allowing the sensor interface capsularattachment layer 15 to be secured thereto. The top member sheath 4 maybe coated with a sheath capsular attachment layer 16; in someembodiments, the sheath capsular attachment layer extends beyond the topmember sheath (e.g., it may jacket the sides of the device or the bottommember).

Maintaining the blood supply near an implanted foreign body like animplanted analyte-monitoring sensor requires stable fixation of FBCtissue on the surface of the foreign body. This can be achieved, forexample, by using capsular attachment membrane materials (e.g., thosematerials that comprise the sensor interface and top member capsularattachment layers) developed to repair or reinforce tissues, including,but not limited to, polyester (DACRON®; DuPont; poly(ethyleneterephthalate)) velour, expanded polytetrafluoroethylene (TEFLON®;Gore), polytetrafluoroethylene felts, polypropylene cloth, and relatedporous implant materials. The preferred material for FBC attachment issurgical-grade polyester velour. FBC tissue tends to aggressively growinto the materials disclosed above and form a strong mechanical bond(i.e., capsular attachment); this fixation of the implant in its capsuleis essential to prevent motion artifact or disturbance of thenewly-developed capillary blood supply. In preferred embodiments,capsular attachment materials are not used in the region of the sensorinterface so as not to interfere with the vasculature development inthat region.

Side braces 3 secure the top member sheath 4 to the bottom member 1 (seeFIG. 1A). A conventional O-ring 7 or other suitable mechanical means maybe used to assist in the attachment of the membrane layers (e.g., theenzyme layer). In a preferred embodiment, the housing is approximately1.4 cm from the base of the bottom member 1 to the top of the sheathcapsular attachment layer 16, and approximately 7.0 cm in length.

The interior (i.e., the cavity) of the housing comprises one or morebatteries 9 operably connected to an electronic circuit means (e.g., acircuit board 8), which, in turn, is operably connected to at least oneelectrode (described below); in preferred embodiments, at least twoelectrodes are carried by the housing. Any electronic circuitry andbatteries that renders reliable, continuous, long-term (e.g., months toyears) results may be used in conjunction with the devices of thepresent invention.

The housing of the devices of the present invention preferably utilize asimple, low-cost packaging technique which protects the components ofthe device for at least one year in aqueous media. In preferredembodiments, the components of the housing (e.g., the top and bottommembers) comprise thermoformed high-density polyethylene. The area inthe cavity of the housing that surrounds the batteries, electroniccircuitry, etc., may be filled with an encapsulant 40 (see FIG. 1A), amaterial that secures in place the components within the cavity but thatdoes not interfere with the operation of those components. In preferredembodiments, the encapsulant 40 is based on mixtures of petroleum waxand low melting temperature resins developed for the hot-melt glueindustry [Shults et al., IEEE Trans. Biomed. Eng. 41:937-942 (1994)]. Inaddition to the high-quality moisture barrier formed with this approach,the electronics (e.g., the circuit board 8) can be recycled by remeltingand draining the encapsulant when the battery expires.

The preferred encapsulant compositions of the present invention compriseapproximately 54% PW 130/35H wax (Astor Wax), approximately 40% MVO 2528resin (Exxon Chemical), and approximately 6% XS 93.04 resin (ExxonChemical, Houston, Tex.). These pelletized compounds render a well-mixedsolution after heating and mixing at about 120° C. for approximately onehour. This solution is then poured into the polyethylene housingcontaining the implant electronics, taking caution to not to exceed theburst temperature of, e.g., approximately 160° C. when lithium batteriesare used.

FIG. 1B depicts a cross-sectional exploded view of the sensor interfacedome 30 of FIG. 1A. Referring to FIG. 1B, the sensor interface domecomprises a region of, for example, epoxy insulation 10 in which isembedded a silver reference electrode 20, a platinum working electrode21, and a platinum counter electrode 22. The present invention isneither limited by the composition of the electrodes nor their positionwithin the sensor interface dome 30.

FIG. 1C depicts a cross-sectional exploded view of theelectrode-membrane region set forth in FIG. 1B detailing the sensor tipand the functional membrane layers. As depicted in FIG. 1C, theelectrode-membrane region comprises several different membrane layers,the compositions and functions of which are described in detail below.The top ends of the electrodes are in contact with the electrolyte phase31, a free-flowing fluid phase. The electrolyte phase is covered by theenzyme membrane 32 that contains an enzyme, e.g., glucose oxidase, andseveral functional polymer layers (as described below). In turn, abioprotective membrane 33 covers the enzyme membrane 32 and serves, inpart, to protect the sensor from external forces that may result inenvironmental stress cracking of the enzyme membrane 32. Finally, anangiogenic layer 34 is placed over the bioprotective membrane 33 andserves to promote vascularization in the sensor interface region.

A retaining gasket 18 composed of, for example, silicone rubber, is usedto retain the sensor interface capsular attachment layer 15 (FIGS. 1A-B)and the angiogenic layer 34 and the bioprotective membrane 33 (notshown). In preferred embodiments, the angiogenic layer 34 and thebioprotective membrane 33 pass over the tip of the sensor interface dome30, over the O-ring 7, and then under the sensor interface capsularattachment layer 15 and the retaining gasket 18.

The present invention contemplates the construction of the membranelayers of the sensor interface region using standard film coatingtechniques. This type of membrane fabrication facilitates control ofmembrane properties and membrane testing.

III. Sensor Interface

As alluded to above and disclosed in FIG. 1C, in a preferred embodiment,the sensor interface region comprises several different layers andmembranes that cover the electrodes of an implantable analyte-measuringdevice. The characteristics of these layers and membranes are nowdiscussed in more detail. The layers and membranes prevent directcontact of the biological fluid sample with the electrodes, whilepermitting selected substances (e.g., analytes) of the fluid to passtherethrough for electrochemical reaction with the electrodes.

The membranes used in the sensor interface region are semipermeablemembranes. Generally speaking, the two fundamental diffusion processesby which a semipermeable membrane can limit the amount of a substancethat passes therethrough are i) diffusion through the semipermeablemembrane as a porous structure and ii) diffusion through thesemipermeable membrane as a monolithic, homogeneous structure. Thepresent invention is not limited by the nature of the semipermeablemembranes used in the sensor interface region.

A semipermeable membrane that comprises a porous structure consists of arelatively impermeable matrix that includes a plurality of “microholes”or pores of molecular dimensions. Transfer through these membranes isprimarily due to passage of substances through the pores (i.e., themembrane acts as a microporous barrier or sieve). Examples of materialsthat may be used to form porous, semipermeable membranes include, butare not limited to, polyethylene, polyvinylchloride,polytetrafluoroethylene, polypropylene, polyacrylamide, celluloseacetate, polymethyl methacrylate, silicone polymers, polycarbonate, andcellulosic polymers.

Because diffusion is primarily due to passage of the substance throughpores, the permeability is related to the effective size of the pores,the membrane thickness, and to the molecular size of the diffusingsubstance. As a result, there is little selectivity in the separation oftwo chemically or structurally related molecules, except when theirmolecular size is approximately the same as the size of the pore; whenthis occurs, forces acting between the substance and the surface of thepore channel may influence the rate of transfer. In addition, the uppersize limit to diffusion is determined by the largest pore diameter, andthe overall diffusion rate depends on the total number of pores.

In contrast, passage of a substance through a monolithic, homogeneousmembrane depends upon selective dissolution and diffusion of thesubstance as a solute through a solid, non-porous film. As used herein,the term “monolithic” means substantially non-porous and having agenerally unbroken surface. The term “homogeneous”, with reference to amembrane, means having substantially uniform characteristics from oneside of the membrane to the other. However, a membrane may haveheterogeneous structural domains, for example, created by using blockcopolymers (i.e., polymers in which different blocks of identicalmonomer units alternate with each other), and still be characterizedfunctionally as homogeneous with respect to its dependence upondissolution rather than sieving to effect separation of substances. Amonolithic membrane can thus be used to selectively separate componentsof a solution on the basis of properties other than the size, shape anddensity of the diffusing substances. Monolithic, homogeneous membranesact as a barrier because of the preferential diffusion therethrough ofsome substance. They may be formed from materials such as thosepreviously listed for porous membranes, including, but not limited to,polyethylene, polyvinylchloride, tetrafluorethylene, polypropylene,polyacrylamide, polymethyl methacrylate, silicone polymers,polycarbonate, collagen, polyurethanes and block copolymers thereof(block copolymers are discussed in U.S. Pat. Nos. 4,803,243 and4,686,044, hereby incorporated by reference).

A. Angiogenic Layer

The term “angiogenic” is employed to describe a material that has thecharacteristic of stimulating the growth of new vascular structures bythe host close to a device. For implantable glucose monitoring devices,a sensor/tissue interface must be created which provides the sensor withoxygen and glucose concentrations comparable to that normally availableto tissue comprised of living cells. Absent such an interface, thesensor is associated with unstable and chaotic performance indicatingthat inadequate oxygen and/or glucose are reaching the sensor. Thedevelopment of suitable interfaces in other contexts has been reported.For example, investigators have developed techniques which stimulate andmaintain blood vessels inside a FBC to provide for the demanding oxygenneeds of pancreatic islets within an implanted membrane. [See, e.g.,Brauker et al., Abstract from 4th World Biomaterials Congress, Berlin(1992)]. These techniques depend, in part, on the use of a vascularizinglayer on the exterior of the implanted membrane. However,previously-described implantable analyte-monitoring devices have notbeen able to successfully maintain sufficient blood flow to the sensorinterface.

As described above, the outermost layer of the electrode-membrane regioncomprises an angiogenic material. The angiogenic layer of the devices ofthe present invention may be constructed of membrane materials such ashydrophilic polyvinylidene fluoride (e.g., Durapore®; Millipore), mixedcellulose esters (e.g., MF; Millipore), polyvinyl chloride (e.g., PVC;Millipore), and other polymers including, but not limited to,polyethylene, polytetrafluoroethylene, cellulose acetate, cellulosenitrate, polycarbonate, nylon, polyester, mixed esters of cellulosepolyvinylidene difluoride, silicone, polyacrylonitrile, polypropylene,polysulphone, and polymethacrylate. Preferably, the thickness of theangiogenic layer is about 10 μm to about 20 μm. The angiogenic layercomprises pores sizes of about 0.5 to about 20 μm, and more preferablyabout 1.0 to about 10 μm, sizes that allow most substances to passthrough, including, e.g., macrophages. The preferred material isexpanded PTFE of a thickness of about 15 μm and pore sizes of about 5 μmto about 10 μm.

To further promote stable foreign body capsule structure withoutinterfering with angiogenesis, an additional outermost layer of materialcomprised of a thin low-density non-woven polyester (e.g., manufacturedby Gore) can be laminated over the preferred PTFE described above. Inpreferred embodiments, the thickness of this layer is about 120 μm. Thisadditional thin layer of material does not interfere with angiogenesisand enhances the manufacturability of the angiogenic layer. [See U.S.Pat. No. 5,453,278 to Chan et al., hereby incorporated by reference; PCTPatent Publication Nos. 96/32076, 96/01611, and 92/07525 assigned toBaxter].

B. Bioprotective Membrane

The inflammatory response that initiates and sustains a FBC isassociated with both advantages and disadvantages. Some inflammatoryresponse is needed to create a new capillary bed in close proximity tothe surface of the sensor in order to i) continuously deliver adequateoxygen and glucose and ii) create sufficient tissue ingrowth to anchorthe implant and prevent motion artifact. On the other hand, inflammationis associated with invasion of tissue macrophages which have the abilityto biodegrade many artificial biomaterials (some of which were, untilrecently, considered nonbiodegradable). When activated by a foreignbody, tissue macrophages degranulate, releasing from their cytoplasmicmyeloperoxidase system hypochlorite (bleach), H₂O₂ and other oxidantspecies. Both hypochlorite and H₂O₂ are known to break down a variety ofpolymers, including polyurethane, by a phenomenon referred to asenvironmental stress cracking. [Phillips et al., J. Biomat. Appl.,3:202-227 (1988); Stokes, J. Biomat. Appl. 3:228-259 (1988)]. Indeed,environmental stress cracking has been shown to limit the lifetime andperformance of an enzyme-active polyurethane membrane stretched over thetip of a glucose sensor. [Updike et al., Am. Soc. Artificial InternalOrgans, 40:157-163 (1994)].

Because both hypochlorite and H₂O₂ are short-lived chemical species invivo, biodegradation will not occur if macrophages are kept a sufficientdistance from the enzyme active membrane. The present inventioncontemplates the use of protective biomaterials of a few micronsthickness or more (i.e., a bioprotective membrane) which are permeableto oxygen and glucose and are placed over the tip of the sensor to keepthe macrophages from gaining proximity to the sensor membrane. Thedevices of the present invention are not limited by the nature of thebioprotective layer. However, the bioprotective layer should bebiostable for long periods of time (e.g., several years); the presentinvention contemplates the use of polymers including, but not limitedto, polypropylene, polysulphone, polytetrafluoroethylene (PTFE), andpoly(ethylene terephthalate) (PET).

Preferably, the bioprotective layer is constructed of expanded PTFE witha pore size of about 0.2 μm to about 0.5 μm and a thickness of about 15to about 35 μm. Most preferably, the bioprotective layer is constructedof expanded PTFE with a pore size of 0.4 μm and a thickness ofapproximately 25 μm (e.g., Millicell CM-Biopore®; Millipore).

C. The Enzyme Membrane

The present invention contemplates membranes impregnated with enzyme. Itis not intended that the present invention be limited by the nature ofthe enzyme membrane. The enzyme membrane of a preferred embodiment isdepicted in FIG. 1C as being a single, homogeneous structure. However,in preferred embodiments, the enzyme membrane comprises a plurality ofdistinct layers. In a particularly preferred embodiment, the enzymemembrane comprises the following four layers (in succession from thebioprotective membrane to the electrolyte phase): i) a resistance layer;ii) an enzyme layer; iii) an interference layer; and iv) an electrolytelayer.

Resistance Layer

There is a molar excess of glucose relative to the amount of oxygen insamples of blood. Indeed, for every free oxygen molecule inextracellular fluid, there are typically more than 100 glucose moleculespresent [Updike et al., Diabetes Care 5:207-21 (1982)]. However, animmobilized enzyme-based sensor using oxygen (O₂) as cofactor must besupplied with oxygen in non-rate-limiting excess in order to respondlinearly to changes in glucose concentration while not responding tochanges in oxygen tension. More specifically, when a glucose-monitoringreaction is oxygen-limited, linearity is not achieved above minimalconcentrations of glucose. Without a semipermeable membrane over theenzyme layer, linear response to glucose levels can be obtained only upto about 40 mg/dL; however, in a clinical setting, linear response toglucose levels are desirable up to at least about 500 mg/dL.

The resistance layer comprises a semipermeable membrane that controlsthe flux of oxygen and glucose to the underlying enzyme layer (i.e.,limits the flux of glucose), rendering the necessary supply of oxygen innon-rate-limiting excess. As a result, the upper limit of linearity ofglucose measurement is extended to a much higher value than that whichcould be achieved without the resistance layer. The devices of thepresent invention contemplate resistance layers comprising polymermembranes with oxygen-to-glucose permeability ratios of approximately200:1; as a result, one-dimensional reactant diffusion is adequate toprovide excess oxygen at all reasonable glucose and oxygenconcentrations found in the subcutaneous matrix [Rhodes et al., Anal.Chem., 66:1520-1529 (1994)].

In preferred embodiments, the resistance layer has a thickness of lessthan about 45 μm, more preferably in the range of about 15 to about 40μm and most preferably in the range of about 20 to about 35 μm.

Enzyme Layer

In addition to glucose oxidase, the present invention contemplates theuse of a membrane layer impregnated with other oxidases, e.g., galactoseoxidase, uricase. For an enzyme-based electrochemical glucose sensor toperform well, the sensor's response must neither be limited by enzymeactivity nor cofactor concentration. Because enzymes, including the veryrobust glucose oxidase, are subject to deactivation as a function ofambient conditions, this behavior needs to be accounted for inconstructing sensors for long-term use.

The principle of losing half of the original enzyme activity in aspecific time may be used in calculating how much enzyme needs to beincluded in the enzyme layer to provide a sensor of required lifetime(see Experimental section). Previously, researchers have found that,when placed in a saline solution at 37° C., glucose electrodes lose halfof their electrode enzyme activity in 85 to 105 days [See, e.g., Tse andGough, Biotechnol. Bioeng. 29:705-713 (1987)]. Under reasonable diabeticconditions and normal enzyme loading (e.g., 2×10⁻⁴ M glucose oxidase;see Example 4), useful sensor lifetimes can last for at least one year.However, exposure of the sensor to high levels of glucose in combinationwith low oxygen levels for prolonged periods can result in shortenedsensor lifetimes. [Rhodes et al., Anal. Chem., 66:1520-1529 (1994)].

Excess glucose oxidase loading is required for long sensor life. TheExperimental section provides a procedure that can be used to determinethe appropriate amount of enzyme to be included in the enzyme layer.When excess glucose oxidase is used, up to two years of performance ispossible from the glucose-monitoring devices contemplated by the presentinvention.

Interference Layer

The interference layer comprises a thin, hydrophobic membrane that isnon-swellable and has a low molecular weight cut-off. The interferencelayer is permeable to relatively low molecular weight substances, suchas hydrogen peroxide, but restricts the passage of higher molecularweight substances, including glucose and ascorbic acid. The interferencelayer serves to allow analytes and other substances that are to bemeasured by the electrodes to pass through, while preventing passage ofother substances.

The interference layer has a preferred thickness of less than about 5μm, more preferably in the range of about 0.1 to about 5 μm and mostpreferably in the range of about 0.5 to about 3 μm.

Electrolyte Layer

To ensure electrochemical reaction, the electrolyte layer comprises asemipermeable coating that maintains hydrophilicity at the electroderegion of the sensor interface. The electrolyte layer enhances thestability of the interference layer of the present invention byprotecting and supporting the membrane that makes up the interferencelayer. Furthermore, the electrolyte layer assists in stabilizingoperation of the device by overcoming electrode start-up problems anddrifting problems caused by inadequate electrolyte. The bufferedelectrolyte solution contained in the electrolyte layer also protectsagainst pH-mediated damage that may result from the formation of a largepH gradient between the hydrophobic interference layer and the electrode(or electrodes) due to the electrochemical activity of the electrode.

Preferably the coating comprises a flexible, water-swellable,substantially solid gel-like film having a “dry film” thickness of about2.5 μm to about 12.5 μm, preferably about 6.0 μm. “Dry film” thicknessrefers to the thickness of a cured film cast from a coating formulationonto the surface of the membrane by standard coating techniques. Thecoating formulation comprises a premix of film-forming polymers and acrosslinking agent and is curable upon the application of moderate heat.

Suitable coatings are formed of a curable copolymer of a urethanepolymer and a hydrophilic film-forming polymer. Particularly preferredcoatings are formed of a polyurethane polymer having anionic carboxylatefunctional groups and non-ionic hydrophilic polyether segments, which iscrosslinked in the present of polyvinylpyrrolidone and cured at amoderate temperature of about 50° C.

Particularly suitable for this purpose are aqueous dispersions offully-reacted colloidal polyurethane polymers having cross-linkablecarboxyl functionality (e.g., BAYBOND®; Mobay Corporation). Thesepolymers are supplied in dispersion grades having apolycarbonate-polyurethane backbone containing carboxylate groupsidentified as XW-121 and XW-123; and a polyester-polyurethane backbonecontaining carboxylate groups, identified as XW-110-2. Particularlypreferred is BAYBOND® 123, an aqueous anionic dispersion of an aliphatepolycarbonate urethane polymer sold as a 35 weight percent solution inwater and co-solvent N-methyl-2-pyrrolidone.

Polyvinylpyrrolidone is also particularly preferred as a hydrophilicwater-soluble polymer and is available commercially in a range ofviscosity grades and average molecular weights ranging from about 18,000to about 500,000, under the PVP K® homopolymer series by BASF Wyandotteand by GAF Corporation. Particularly preferred is the homopolymer havingan average molecular weight of about 360,000 identified as PVP-K90 (BASFWyandotte). Also suitable are hydrophilic, film-forming copolymers ofN-vinylpyrrolidone, such as a copolymer of N-vinylpyrrolidone and vinylacetate, a copolymer of N-vinylpyrrolidone, ethylmethacrylate andmethacrylic acid monomers, and the like.

The polyurethane polymer is crosslinked in the presence of thepolyvinylpyrrolidone by preparing a premix of the polymers and adding across-linking agent just prior to the production of the membrane.Suitable cross-linking agents can be carbodiimides, epoxides andmelamine/formaldehyde resins. Carbodiimide is preferred, and a preferredcarbodiimide crosslinker is UCARLNK® XL-25 (Union Carbide).

The flexibility and hardness of the coating can be varied as desired byvarying the dry weight solids of the components in the coatingformulation. The term “dry weight solids” refers to the dry weightpercent based on the total coating composition after the time thecrosslinker is included. A preferred useful coating formulation cancontain about 6 to about 20 dry weight percent, preferably about 8 dryweight percent, polyvinylpyrrolidone; about 3 to about 10 dry weightpercent preferably about 5 dry weight percent cross-linking agent; andabout 70 to about 91 weight percent, preferably about 87 weight percentof a polyurethane polymer, preferably a polycarbonate-polyurethanepolymer. The reaction product of such a coating formulation is referredto herein as a water-swellable cross-linked matrix of polyurethane andpolyvinylpyrrolidone.

D. The Electrolyte Phase

The electrolyte phase is a free-fluid phase comprising a solutioncontaining at least one compound, usually a soluble chloride salt, thatconducts electric current. The electrolyte phase flows over theelectrodes (see FIG. 1C) and is in contact with the electrolyte layer ofthe enzyme membrane. The devices of the present invention contemplatethe use of any suitable electrolyte solution, including standard,commercially available solutions.

Generally speaking, the electrolyte phase should have the same or lessosmotic pressure than the sample being analyzed. In preferredembodiments of the present invention, the electrolyte phase comprisesnormal saline.

E. Electrode

The electrode assembly of this invention may also be used in the mannercommonly employed in the making of amperometric measurements. A sampleof the fluid being analyzed is placed in contact with a referenceelectrode, e.g., silver/silver-chloride, and the electrode of thisinvention which is preferably formed of platinum. The electrodes areconnected to a galvanometer or polarographic instrument and the currentis read or recorded upon application of the desired D.C. bias voltagebetween the electrodes.

The ability of the present device electrode assembly to accuratelymeasure the concentration of substances such as glucose over a broadrange of concentrations in fluids including undiluted whole bloodsamples enables the rapid and accurate determination of theconcentration of those substances. That information can be employed inthe study and control of metabolic disorders including diabetes.

IV. Sensor Implantation and Radiotelemetric Output

Long-term sensor performance is best achieved, and transcutaneousbacterial infection is eliminated, with implanted devices capable ofradiotelemetric output. The present invention contemplates the use ofradiotelemetry to provide data regarding blood glucose levels, trends,and the like. The term “radiotelemetry” refers to the transmission byradio waves of the data recorded by the implanted device to an ex vivorecording station (e.g., a computer), where the data is recorded and, ifdesired, further processed.

Although totally implanted glucose sensors of three month lifetime, withradiotelemetric output, have been tested in animal models at intravenoussites [see, e.g. Armour et al., Diabetes, 39:1519-1526 (1990)],subcutaneous implantation is the preferred mode of implantation [see,e.g., Gilligan et al., Diabetes Care 17:882-887 (1994)]. Thesubcutaneous site has the advantage of lowering the risk forthrombophlebitis with hematogenous spread of infection and also lowersthe risk of venous thrombosis with pulmonary embolism. In addition,subcutaneous placement is technically easier and more cost-effectivethan intravenous placement, as it may be performed under localanesthesia by a non-surgeon health care provider in an outpatientsetting.

Preferably, the radiotelemetry devices contemplated for use inconjunction with the present invention possess features including smallpackage size, adequate battery life, acceptable noise-free transmissionrange, freedom from electrical interference, and easy data collectionand processing. Radiotelemetry provides several advantages, one of themost important of which is the ability of an implanted device to measureanalyte levels in a sealed-off, sterile environment.

The present invention is not limited by the nature of the radiotelemetryequipment or methods for its use. Indeed, commercially availableequipment can be modified for use with the devices of the presentinvention (e.g., devices manufactured by Data Sciences). Similarly,custom-designed radiotelemetry devices like those reported in theliterature can be used in conjunction with the implantableanalyte-measuring devices of the present invention [see, e.g., McKeanand Gough, IEEE Trans. Biomed. Eng. 35:526-532 (1988); Shichiri et al.,Diabetes Care 9:298-301 (1986); and Shults et al., IEEE Trans. Biomed.Eng. 41:937-942 (1994)]. In a preferred embodiment, transmitters areprogrammed with an external magnet to transmit at 4-, 32-, or 256-secondintervals depending on the need of the subject; presently, batterylifetimes at the current longest transmission intervals (about 256seconds) is approximately up to two years.

V. Response Time and Calibration

Every measurement method reports data with some delay after the measuredevent. For data to be useful, this delay must be smaller than some timedepending on the needs of the method. Thus, response time of the currentinvention has been carefully studied. The use of the term “initialresponse” is not to be confused with the term “response time.” After astep function change in glucose concentration, the time delay before thefirst unequivocal change in sensor signal occurs is the “initialresponse,” while the following time delay to reach 90% of thesteady-state signal development is the “response time.” “Response time”is the factor which normally controls how quickly a sensor can track adynamically changing system.

Furthermore, the time required before a glucose sensor in a FBC willindicate an initial response to a bolus intravenous glucose injection isa function of the animal “circulation time” and the sensor's “initialresponse”. The circulation time is the time required for a bolus glucoseinjection to reach the site of sensor implantation.

Generally speaking, equilibration between vascular and interstitialcompartments for glucose is so rapid that it plays no role in either theinitial response or the observed response time. If the tip of the sensoris in intimate contact with the interstitial compartment (e.g., FBC),then there is no significant delay in glucose diffusing from thecapillary lumen to the tip of the sensor. The inventors have found thatthe glucose sensors of the present invention provide initial responsesof about 30 seconds in dogs about half of which is circulation time. Thedog model represents a useful and accepted model for determining theefficacy of glucose monitoring devices.

While the devices of the present invention do not require a specificresponse time, in preferred embodiments of the present invention, the invitro 90% response times at 37° C. for subsequently subcutaneouslyimplanted devices are in the range of 2 to 5 minutes in dogs. Though theuse of the devices of the present invention does not require anunderstanding of the factors that influence response time or thefactors' mechanisms of action, in vivo response times are believed to beprimarily a function of glucose diffusion through the sensor membrane(e.g., a 40-60 micron membrane). Of note, response times of up to about10 minutes do not limit the clinical utility of tracking blood glucosein diabetic patients because physiologic or pathologic glucose levels donot change more rapidly than a few percent per minute.

In calibrating the glucose sensors of the present invention, a singlepoint recalibration of the sensor at four-week intervals against anacceptable glucose reference method is preferred (e.g., calibrationagainst blood obtained from a finger-prick). Generally speaking, therecalibration amounts to a simple adjustment in sensor gain. The sensoroffset current (i.e., the current at 0 mg/dL glucose) needs to remaininvariant over the duration of the implant for the sensor to provideoptimal data.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the preceding description and the experimental disclosure whichfollows, the following abbreviations apply: Eq and Eqs (equivalents);mEq (milliequivalents); M (molar); mM (millimolar) μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); Kg(kilograms); L (liters); mL (milliliters); dL (deciliters); μL(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); h and hr (hours); min. (minutes); s and sec. (seconds); °C. (degrees Centigrade); Astor Wax (Titusville, Pa.); BASF WyandotteCorporation (Parsippany, N.J.); Data Sciences, Inc. (St. Paul, Minn.);DuPont (DuPont Co., Wilmington, Del.); Exxon Chemical (Houston, Tex.);GAF Corporation (New York, N.Y.); Markwell Medical (Racine, Wis.);Meadox Medical, Inc. (Oakland, N.J.); Mobay (Mobay Corporation,Pittsburgh, Pa.); Sandoz (East Hanover, N.J.); and Union Carbide (UnionCarbide Corporation; Chicago, Ill.).

Example 1

The polyurethanes are preferably prepared as block copolymers bysolution polymerization techniques as generally described in Lyman [J.Polymer Sci. 45:49 (1960)]. Specifically, a two-step solutionpolymerization technique is used in which the poly(oxyethylene) glycolis first “capped” by reaction with a diisocyanate to form amacrodiisocyanate. The macrodiisocyanate is then coupled with a diol (ordiamine) and the diisocyanate to form a block copolyetherurethane (or ablock copolyurethaneurea). The resulting block copolymers are tough andelastic and may be solution-cast in N,N-dimethylformamide to yield clearfilms that demonstrate good wet strength when swollen in water.

In particular, a mixture of 8.4 g (0.006 mol), poly(oxyethylene) glycol(CARBOWAX® 1540, Union Carbide), and 3.0 g (0.012 mol)4,4′-diphenylmethane diisocyanate in 20 mL dimethylsulfoxide/4-methyl-2-pentanone (50/50) is placed in a three-necked flaskequipped with a stirrer and condenser and protected from moisture. Thereaction mixture is stirred and heated at 110° C. for about one hour. Tothis clear solution is added 1.5 g (0.014 mol) 1,5-pentanediol and 2.0 g(0.008 mol) 4,4′-diphenylmethane diisocyanate.

After heating at 110° C. for an additional two hours, the resultingviscous solution is poured into water. The tough, rubbery, white polymerprecipitate that forms is chopped in a Waring Blender, washed with waterand dried in a vacuum oven at about 60° C. The yield is essentiallyquantitative. The inherent viscosity of the copolymer in N,N-dimethylformamide is 0.59 at 30° C. (at a concentration of about 0.05 percent byweight).

Example 2

As previously described, the electrolyte layer, the membrane layerclosest to the electrode, can be coated as a water-swellable film. Thisexample illustrates a coating comprising a polyurethane having anioniccarboxylate functional groups and hydrophilic polyether groups andpolyvinylpyrrolidone (PVP) that can be cross-linked by carbodiimide.

A coating preparation is prepared comprising a premix of a colloidalaqueous dispersion of particles of a urethane polymer having apolycarbonate-polyurethane (PC-PU) backbone containing carboxylategroups and the water-soluble hydrophilic polymer, PVP, which iscrosslinked by the addition of the cross-linking agent just beforeproduction of the coated membrane. Example coating formulations areillustrated in Table 1.

TABLE 1 A B C Dry Dry Dry Weight Weight Weight % % % Weight SolidsWeight Solids Weight Solids Premix PVP¹ 48 6 64 8 160 20 PC-PV² 260 91248 87 200 70 Cross-Linking Agent Carbodiimide³ 6 3 10 5 20 10 Totals314 100 322 100 380 100

-   1. Aqueous solution containing 12.5 weight percent PVP prepared from    polyvinylpyrrolidone having a number average molecular weight of    about 360,000 sold as a powder under the trademark BASF K90 by BASF    Wyandotte Corporation.-   2. Colloidal dispersion of a polycarbonatepolyurethane (PCPU)    polymer at about 35 weight percent solids in a co-solvent mixture of    about 53 weight percent water and about 12 weight percent    N-methyl-2-pyrrolidone (BAYBOND® 123 or XW 123; Mobay Corporation).    As supplied, the dispersion has a pH of about 7.5-9.0.-   3. Carbodiimide (UCARLNK® XL25SE, Union Carbide Corporation)    supplied at about 50 weight percent solids in a solvent solution of    propylene glycol monomethylether acetate.

The viscosity and pH of the premix can be controlled and maintainedduring processing and to prolong its useful life by adding water oradjusting the pH with dilute ammonia solution or an equivalent baseprior to adding the crosslinker.

For production, the coating is applied with a Mayer rod onto the unboundsurface of a multilayered membrane. The amount of coating applied shouldcast a film having a “dry film” thickness of about 2.5 μm to about 12.5μm, preferably about 6.0 μm. The coating is dried above room temperaturepreferably at about 50° C. This coating dries to a substantially solidgel-like film that is water swellable to maintain electrolyte betweenthe membrane covering the electrode and the electrode in the electrodeassembly during use.

Example 3

The following procedure was used to determine the amount of enzyme to beincluded in the enzyme layer. It is to be understood that the presentinvention is not limited to the use of this or a similar procedure, butrather contemplates the use of other techniques known in the art.

A starting glucose oxidase concentration of 2×10⁻⁴ M was calculated fromthe enzyme weight and the final volume of the enzyme layer. Thereafter,a series of eight additional membrane formulations was prepared bydecrementing enzyme concentration in 50% steps (referred to as a changeof one “half loading”) down to 7.8×10⁻⁷ M. Sensor responses were thencollected for this range of enzyme loadings and compared tocomputer-simulated sensor outputs. The simulation parameter set usedincluded previously-determined membrane permeabilities and theliterature mechanisms and kinetics for glucose oxidase. [Rhodes et al.,Anal. Chem., 66:1520-1529 (1994)].

There was a good match of real-to-simulated sensor output at allloadings (data not shown). Approximately a six-to-seven “half loading”drop in enzyme activity was required before the sensor output dropped10%; another two-to-three half loading drop in enzyme activity wasrequired to drop the sensor response to 50% of the fully loaded sensorresponse. These results indicate that, at the loading used and the decayrates measured, up to two years of performance is possible from thesesensors when the sensor does not see extended periods of high glucoseand physiologically low O₂ concentrations.

Example 4

This example illustrates long-term glucose sensor device responsefollowing subcutaneous implantation of sensor devices contemplated bythe present invention into a dog. The stages of FBC development areindicated by the long term glucose sensor device response.

FIG. 2 graphically depicts glucose levels as a function of the number ofdays post-implant. The data in FIG. 2 was taken at four-minute intervalsfor 60 days after implantation. Sensor response is calculated from asingle preimplant calibration at 37° C. Normal canine fasting glucoseconcentration of 5.5 mM is shown for comparison.

The data set forth in FIG. 2 can be used to illustrate the fourtypically identifiable phases in FBC formation. Phase 1 shows rapidlydropping response from the time of implant to, in this case, day 3.Though an understanding of the mechanism for this drop in sensor outputis not required in order to practice the present invention, it isbelieved to reflect low pO₂ and low glucose present in fluid contactingthe sensor. Phase 2 shows intermittent sensor-tissue contact in seromafluid from, in this case, day 3 to about day 13. During this phase,fragile new tissue and blood supply intermittently make contact with thesensor (which is surrounded by seroma fluid). Phase 3 showsstabilization of capillary supply between, in this case, days 13 and 22.More specifically, the noise disappears and sensor output rises overapproximately six days to a long term level associated with tracking ofFBC glucose. Again, though an understanding of this effect is notrequired to practice the present invention, the effect is believed toreflect consistent contact of FBC tissue with the sensor surface. Phase4 from, in this case, day 22 to day 60, shows duration of useful sensordevice life. While there are timing variations of the stages from sensordevice to sensor device, generally speaking, the first three steps ofthis process take from 3 days to three weeks and continuous sensing hasbeen observed for periods thereafter (e.g., for periods of 150 days andbeyond).

Example 5

In addition to collecting normoglycemic or non-diabetic dog data fromthe sensor of the present invention as shown in Example 4, calibrationstability, dynamic range, freedom from oxygen dependence, response timeand linearity of the sensor can be studied by artificial manipulation ofthe intravenous glucose of the sensor host.

This was done in this example via infusion of a 15 g bolus of 50%sterile Dextrose given intravenously in less than about 20 seconds.Reference blood glucose data was then taken from a different vein at 2-5minute intervals for up to 2 hours after bolus infusion. FIG. 3 depictscorrelation plots of six bolus infusion studies, at intervals of 7-10days on one sensor of the present invention. Sensor glucoseconcentrations are calculated using a single 37° C. in vitropreimplantation calibration. The sensor response time is accounted forin calculating the sensor glucose concentrations at times of referenceblood sampling by time shifting the sensor data 4 minutes.

As with any analytical system, periodic calibration should be performedwith the devices of the present invention. Thus, the present inventioncontemplates some interval of calibration and/or control testing to meetanalytical, clinical and regulatory requirements.

Example 6

This example describes experiments directed at sensor accuracy andlong-term glucose sensor response of several sensor devices contemplatedby the present invention.

Pre-Implant In Vitro Evaluation

In vitro testing of the sensor devices was accomplished in a mannersimilar to that previously described. [Gilligan et al., Diabetes Care17:882-887 (1994)]. Briefly, sensor performance was verified bydemonstrating linearity to 100 mg/dL glucose concentration steps from 0mg/dL through 400 mg/dL (22 mM) with a 90% time response to the glucosesteps of less than 5 minutes. A typical satisfactory response to thisprotocol is shown in FIG. 4. Modulating dissolved oxygen concentrationfrom a pO₂ of 150 down to 30 mm Hg (0.25 to 0.05 mM) showed no more thana 10% drop in sensor output at 400 mg/dL for the preferred sensordevices of the present invention. Stability of calibration wasmaintained within 10% for one week before the final bioprotective andangiogenesis membranes were added to finalize the implant package. Afinal calibration check was made and had to be within 20% of the priorresults for the sensor to be passed on to the implant stage. These finalcalibration factors (linear least squares regression for the zeroglucose current and output to 100 mg/dL current) are used for theinitial in vivo calibration. Sensor devices were then wet sterilizedwith 0.05% thimerosal for 24 hours just prior to implantation.

In Vivo Testing

Six sensor devices meeting the parameters described above weresurgically implanted under general anesthesia (pentothal induction toeffect, followed by halothane maintenance) into the paravertebralsubcutaneous tissue of the same mongrel non-diabetic dog. A two-inchskin incision was made several inches from the spine for each implantallowing the creation of a tight-fitting subcutaneous pouch by bluntdissection. The implant was then inserted into the pouch in sensor-downconfiguration. Subcutaneous tissue was then closed with 3-0 vicryl andskin with 2-0 nylon. Animals were closely monitored for discomfort aftersurgery and analgesics administered if necessary.

These sensor devices were implanted two-at-a-time in the same dog atapproximately six week intervals. Four of the sensor devices werecovered with a PTFE-comprising angiogenic layer (these sensor deviceswere designated Sensors 1901, 1902, 1903, and 1905), while two of thesensor devices served as control sensor devices and did not contain anangiogenic layer, i.e., they contained a bioprotective membrane and theunderlying sensor interface structures, as previously described (thesesensor devices were designated Sensors 1904 and 1906). To insureanchoring of the device into the subcutaneous tissue, the sensor-side ofeach implant, except for just over the tip of the sensor, was jacketedin surgical grade double velour polyester fabric (Meadox Medical, Inc.).All sensor devices were tracked after implantation at four-minuteintervals using radiotelemetry to follow the long-term sensor responseto normoglycemia, allowing verification of the long-term stability ofthe sensors. To screen for sensor response to changing glucose onselected days following implantation, the response to 0.5 mg glucagonadministered subcutaneously was assessed. Responding sensors wereidentified by a characteristically stable signal prior to glucagonadministration followed by a substantial increase in signal within 20minutes of glucagon injection. The sensor transients then reversed andreturned to the prior signal levels within one hour after glucagoninjection.

To determine in vivo sensor response times, short-term stability,linearity to glucose concentration, and possible oxygen cofactorlimitation effects, glucose infusion studies of up to five hoursduration were performed on the dog. These studies were run approximatelyonce every three weeks. The dog was pretrained to rest comfortably andwas fully alert during this testing. These experiments used thesomatostatin analog octreotide (SANDOSTATIN®, Sandoz) to inhibit therelease of insulin, allowing for a slow ramping of blood glucose to the400-500 mg/dL concentration range.

Sensors were monitored at 32-second intervals to allow simultaneoustracking of up to six sensor devices. In this protocol, octreotide wasinjected (36-50 μg/kg) 15-20 minutes before initiation of the glucoseinfusion. Two peripheral veins were cannulated in the dog to allow forglucose infusion and blood glucose sampling. Ten percent dextrose (0.55mM) was continuously infused at gradually increasing rates to providesmooth increases in blood glucose from the approximate fasting glucoseconcentration of about 100 mg/dL to greater than 400 mg/dL. Thisinfusion protocol provides sensor glucose concentration data which canbe correlated with reference plasma glucose values when blood sampleswere drawn from the animal every 5-to-10 minutes. The primary referenceglucose determinations were made using a hexokinase method on the DuPontDimension AR®. A DIRECT 30/30® meter (Markwell Medical) was also usedduring the course of the experiment to serve as a secondary monitor forthe reference blood glucose values and estimate when 400 mg/dL had beenreached. At this point the glucose infusion pump was turned off and theblood glucose allowed to return to its normal level.

An additional variation of the protocol described above involvedstudying the effects of insulin administration on blood glucoseconcentration prior to the octreotide injection. For these studies 5units of insulin were injected intravenously, the blood glucose trackeddown to 40 mg/dl with the DIRECT 30/30® (Markwell Medical), theoctreotide injection made as before, and the infusion pump then started.While the initial glucose pump rate was the same, it was increasedfaster than before to counteract the insulin and to maintain the sameexperimental timing.

Once studies were completed, the data was initially analyzed using thefinal in vitro sensor calibration factors to calculate the implantedsensor glucose concentration. If changes were needed in these factors tooptimize the linear regression of sensor to reference blood glucose theywere made and noted and followed over the lifetime of the sensor device.

At varying points in time, the implanted sensor devices became less thanoptimal and were then explanted to determine the underlying cause (lessthan optimal was defined as the inability to accurately track glucoseinfusion during two successive tests). Explantation surgical protocolswere very similar to those used in the implantation procedure exceptthat the foreign body capsule was opened around the perimeter of theoval implant. The back and sides of the housing had no tissue attachment(as they were not covered with polyester velour), and thus easilyseparated from the surrounding tissue. The top of the sensor device withattached capsule was then carefully cut free from the subcutaneoustissues.

Once explanted, the sensor devices were carefully examined under adissecting microscope to look at the state of the capsule tissuecontacting the sensor membranes. Once this had been characterized anddocumented, the tissue was carefully removed from the membrane surfaceand saved for histological examination. If sensor visualizationdemonstrated intact membrane layers an initial in vitro calibrationcheck was performed. The sensors were then disassembled from the topmembrane down (i.e., from the membrane furthest from the electrodes)with a glucose and hydrogen peroxide calibration check made afterremoval of each layer. This allowed differentiation of the mechanismsleading to less than optimal results in the membranes and determinationof whether processes such as environmental stress cracking, biofouling,or loss of enzyme activity were occurring.

Results and Conclusions

Typical Glucose Infusion Studies: The six sensor devices were trackedfor 20-150 days and were evaluated using the octreotide-glucose infusionprotocol. FIGS. 5A, 5B, and 5C graphically depict three in vivo sensorresponse curves (using best case calibration factors) plotted inconjunction with the reference blood glucose values for Sensor 1903 atpost-implant times of 25, 88, and 109 days; this data is representativeof the data obtainable with the sensor devices of the present invention.Referring to FIGS. 5A-C, the arrow labelled “#1” indicates octreotideinjection, the arrow labelled “#2” indicates the turning on of theglucose infusion pump, and the arrow labelled “#3” indicates the turningoff of this pump. The 90% response time for this sensor over itslifetime ranged from 5-to-10 minutes and was 5 minutes for the datashown. Such time responses are adequate, since changes in diabeticpatients occur at slower rates than used with infusion protocols.

FIG. 6 graphically depicts sensor glucose versus reference glucose (forSensor 1903) using the single set of calibration factors from day 88. Asdepicted in FIG. 6, when sensor glucose is plotted versus referenceglucose, the changes in sensor calibration over the lifetime of thesensor become apparent. These changes are reflected primarily in theoutput sensitivity to a known glucose concentration step while the zerocurrent remained quite stable. The results suggest that in vivorecalibration every month would be preferred for this sensor to provideoptimal glucose tracking.

Performance Comparisons

Angiogenesis Stimulating Membrane Sensors Vs. Control Membrane Sensors:

Generally speaking, demonstration of improvement in a sensor can bejudged by noting whether significant improvements in sensor start uptime, increased yields of operating glucose sensors, extension of sensorlifetimes, and maintenance of calibration factors occurs. The lifetimeof a glucose sensor can be defined as the time of first glucose sensing(in this case during a glucagon challenge) to the last glucose infusionstudy which provides correct glucose trends to concentration changes.All sensors showed glucose tracking and only one sensor showed aduration of less than 10 days. Average sensor lifetimes of 84±55 dayswere observed with the sensors containing the angiogenesis-stimulatingmembrane, values superior to the control sensors which only showedlifetimes of 35±10 days. In addition, one of the sensors incorporatingthe angiogenic membrane provided optimal data to 150 days.

The description and experimental materials presented above are intendedto be illustrative of the present invention while not limiting the scopethereof. It will be apparent to those skilled in the art that variationsand modifications can be made without departing from the spirit andscope of the present invention.

We claim:
 1. A device for measuring a glucose concentration in a host,the device comprising: a sensor operably connected to an electroniccircuit and configured to continuously measure a signal associated witha glucose concentration in a host; and a membrane located over at leasta portion of the sensor, wherein the membrane is configured to control aflux of oxygen and glucose, wherein the membrane is configured to useoxygen from a biological fluid surrounding the membrane to catalyze areaction of glucose and oxygen, and wherein the membrane comprises asilicone-containing polymer; wherein the device is capable ofexhibiting, at a glucose concentration of 400 mg/dL, no more than a 10%drop in sensor output over a range of pO₂ from about 150 mm Hg down toabout 30 mm Hg.
 2. The device of claim 1, wherein the membrane comprisesa layer comprising an enzyme.
 3. The device of claim 1, wherein themembrane is monolithic and homogeneous.
 4. The device of claim 1,wherein the membrane is monolithic and heterogeneous.
 5. The device ofclaim 1, wherein the membrane has a thickness of from about 15 micronsto about 60 microns.
 6. The device of claim 1, wherein at least 95% ofglucose concentration values measured by the device are within 25% ofcorresponding values determined by analysis of blood over a useful lifeof the device.
 7. The device of claim 6, wherein the useful life of thedevice is at least 3 days.
 8. The device of claim 1, wherein the deviceis configured to respond substantially linearly to changes in glucoseconcentration at a glucose concentration of up to about 500 mg/dL. 9.The device of claim 1, wherein the membrane comprises an interferencelayer.
 10. The device of claim 6, wherein the useful life of the deviceis at least 1 day.
 11. The device of claim 6, wherein the useful life ofthe device is at least 2 days.
 12. The device of claim 6, wherein theuseful life of the device is at least 4 days.
 13. The device of claim 6,wherein the useful life of the device is at least 5 days.
 14. The deviceof claim 6, wherein the useful life of the device is at least 6 days.15. The device of claim 6, wherein the useful life of the device is atleast 7 days.
 16. The device of claim 6, wherein the useful life of thedevice is at least 10 days.
 17. The device of claim 6, wherein theuseful life is defined by a period of time after stabilization of thedevice.
 18. The device of claim 17, wherein the useful life is furtherdefined by a period of time during which stability of calibration ismaintained.
 19. The device of claim 1, wherein the membrane comprises aurethane polymer or polyurethane.
 20. The device of claim 19, whereinthe urethane polymer or polyurethane comprises apolycarbonate-polyurethane backbone.
 21. The device of claim 1, whereinthe membrane comprises a cross-linked polymer.
 22. The device of claim1, wherein the device is capable of obtaining a calibration stabilitythat is maintained within 10% for one week.
 23. The device of claim 1,wherein the device is configured to respond substantially linearly tochanges in glucose concentration at a glucose concentration of up to atleast 400 mg/dL.
 24. The device of claim 1, wherein the device iscapable of attaining a 90% time response to a 100 mg/dL glucoseconcentration step in less than 5 minutes.
 25. The device of claim 1,wherein the device is configured to reduce or eliminate motion artifact.26. A device for measuring a glucose concentration in a host, the devicecomprising: a sensor operably connected to an electronic circuit andconfigured to continuously measure a signal associated with a glucoseconcentration in a host; and a membrane located over at least a portionof the sensor, wherein the membrane is configured to control a flux ofoxygen and glucose; wherein at least 95% of glucose concentration valuesmeasured by the device are within 25% of corresponding values determinedby analysis of blood over a time period of at least two days.
 27. Thedevice of claim 26, wherein the membrane comprises a layer comprising anenzyme.
 28. The device of claim 26, wherein the membrane is monolithicand homogeneous.
 29. The device of claim 26, wherein the membrane ismonolithic and heterogeneous.
 30. The device of claim 26, wherein themembrane has a thickness of from about 15 microns to about 60 microns.31. The device of claim 26, wherein the time period is at least 3 days.device is at least 3 days.
 32. The device of claim 26, wherein thedevice is capable of exhibiting, at a glucose concentration of 400mg/dL, no more than a 10% drop in sensor output over a range of pO₂ fromabout 150 mm Hg down to about 30 mm Hg.
 33. The device of claim 26,wherein the device is configured to respond substantially linearly tochanges in glucose concentration at a glucose concentration of up toabout 500 mg/dL.
 34. The device of claim 26, wherein the membranecomprises an interference layer.
 35. The device of claim 26, wherein thetime period is at least 1 day.
 36. The device of claim 27, wherein thetime period is at least 2 days.
 37. The device of claim 28, wherein thetime period is at least 4 days.
 38. The device of claim 29, wherein thetime period is at least 5 days.
 39. The device of claim 30, wherein thetime period is at least 6 days.
 40. The device of claim 31, wherein thetime period is at least 7 days.
 41. The device of claim 32, wherein thetime period is at least 10 days.
 42. The device of claim 33, wherein thetime period is defined by a period of time after stabilization of thedevice.
 43. The device of claim 42, wherein the time period is furtherdefined by a period of time during which stability of calibration ismaintained.
 44. The device of claim 34, wherein the time period isdefined by a period of time after stabilization of the device.
 45. Thedevice of claim 44, wherein the time period is further defined by aperiod of time during which stability of calibration is maintained. 46.The device of claim 26, wherein the membrane comprises a urethanepolymer or polyurethane.
 47. The device of claim 46, wherein theurethane polymer or polyurethane comprises a polycarbonate-polyurethanebackbone.
 48. The device of claim 26, wherein the membrane comprises across-linked polymer.
 49. The device of claim 26, wherein the device iscapable of obtaining a calibration stability that is maintained within10% for one week.
 50. The device of claim 26, wherein the device isconfigured to respond substantially linearly to changes in glucoseconcentration at a glucose concentration of up to at least 400 mg/dL.51. The device of claim 26, wherein the device is capable of attaining a90% time response to a 100 mg/dL glucose concentration step in less than5 minutes.
 52. The device of claim 26, wherein the device is configuredto reduce or eliminate motion artifact.
 53. A device for measuring aglucose concentration in a host, the device comprising: an electrodesurface operably connected to an electronic circuit and configured tocontinuously measure in vivo a signal associated with a glucoseconcentration in a host; and a membrane located over at least a portionof the electrode surface, wherein the membrane is configured to controla flux of oxygen and glucose; wherein at least 95% of glucoseconcentration values measured by the device are within 25% ofcorresponding values determined by analysis of blood over a time periodof at least 2 days.
 54. The device of claim 53, wherein the time periodis at least 3 days.
 55. The device of claim 53, wherein the time periodis at least 4 days.
 56. The device of claim 53, wherein the time periodis at least 5 days.
 57. The device of claim 53, wherein the time periodis at least 6 days.
 58. The device of claim 53, wherein the time periodis at least 7 days.
 59. The device of claim 53, wherein the time periodis at least 10 days.
 60. The device of claim 53, wherein the membranecomprises a urethane polymer or polyurethane.
 61. The device of claim60, wherein the urethane polymer or polyurethane comprises apolycarbonate-polyurethane backbone.
 62. The device of claim 53, whereinthe membrane comprises a cross-linked polymer.
 63. The device of claim53, wherein the device is capable of obtaining a calibration stabilitythat is maintained within 10% for one week.
 64. The device of claim 53,wherein the device is configured to respond substantially linearly tochanges in glucose concentration at a glucose concentration of up to atleast 400 mg/dL.
 65. The device of claim 53, wherein the device iscapable of attaining a 90% time response to a 100 mg/dL glucoseconcentration step in less than 5 minutes.
 66. The device of claim 53,wherein the device is configured to reduce or eliminate motion artifact.